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Objectives: Idiopathic inflammatory myopathies (IIM) are a group of refractory systemic autoimmune diseases with unknown pathogenesis, including dermatomyositis (DM) and polymyositis (PM). DNA methylation is an important epigenetic mechanism to regulate gene expression, which plays important roles in IIM. Integrated analysis of the whole-genome DNA methylation and gene expression profiling will provide a comprehensive understanding of the pathogenesis of IIM. Methods: We performed whole-genome DNA methylation and transcription analysis in peripheral blood of 16 DM patients, 8 PM patients and 16 normal controls (NC) using Illumina Human Methyation 450K Beadchips and Illumina Human HT-12 v4.0 Expression Beadchips. Luminex multiplex assay was used to quantify 27 cytokine/chemokine in sera of 30 DM, 16 PM and 237 NC. Genome Studio was used to identify differential DNA methylation and mRNA transcription. Integrative analysis of DNA methylome, mRNA transcriptome and serum cytokines were performed using R, Perl and Circos software. Bioinformatic analysis were performed using DAVID and R. Real-time PCR, Bisulfite pyrosequencing and immunofluorescence were used for validation. Results: Genome-wide DNA methylation analysis identified 241 differential methylated CpG sites (74 hypermethylated and 167 hypomethylated) in DM, and 500 differential methylated CpG sites (153 hypermethylated and 347 hypomethylated) in PM. Transcriptome analysis identified 540 differential expressed genes (277 upregulated and 263 downregulated ) in DM and 242 differential expressed genes (181 upregulated and 61 downregulated) in PM. Integrated analysis of DNA methylome with transcriptome suggested a significant inverse-correlation between DNA methylation and gene expression. 93 upregulated genes associated with hypomethylated CpG sites while 176 down-regulated genes related with hypermethylated CpG sites both DM and PM. Analysis of the DNA methylation and mRNA transcription data identified differential methylated CpG sites and differential expressed genes which are shared and/or unique between DM and PM. Bioinformatics analysis suggested that dysregulated genes which are shared between DM and PM participated in common biologic process, such as immune response, inflammatory response and defense response. However, differential genes which are unique in DM and PM were found to be enriched in type I interferon signaling pathway and cytokine pathway, respectively. 18 interferon related genes were upregulated and hypomethylated, IL-5 was increased and hypomethylated, PDGF-BB was decreased and hypermethylated both in DM and PM. Real-time PCR and bisulfite pyrosequencing validated 13 top changed methylation sites and genes, and showed significant correlation between DNA methylation status and gene expression in most genes. Conclusions: Our study identified differentially methylated CpG sites which have potential regulatory effects on differential gene expression in DM and PM. We also revealed dysregulated CpG sites and genes involved in common and specific biologic process in DM and PM. Type I interferon signaling pathway was significantly enrichment in DM while Cytokine signaling pathway was significantly enrichment in PM. Our observations have laid the groundwork for further diagnostic and mechanistic studies of DM and PM.

Studying the pathogenesis of neurological diseases with animal models might not always truly recapitulate their pathophysiology, due to species differences. Fortunately, human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), especially derived from patients, have been widely employed to induce neural progenitor cells (NPCs) and further multiple neural subtypes. Particularly in the past decade, hPSC-based cell sources have been applied in studying neural development, cell therapy, disease modeling, and drug screening, among others. The generation of unlimited amount of neurons also facilitates a variety of biochemical assays, mass spectrometry, omic analysis, and next-generation sequencing, which thus provides an excellent tool in modeling neurodegenerative and neurodevelopmental diseases. Dysfunction or death of motor neurons (MNs) in the spinal cord and motor cortex is implicated in various motor neuron diseases (MNDs). Yet, producing high-purity and high-yield MNs remains a major challenge due to the complexity of MN specification during development. In this chapter, we describe a method of generating functional MNs via lentiviral delivery of transcription factors, based on the preservable NPC platform derived from hPSCs. Specifically, we transduce NPCs with a single lentivirus co-expressing three transcription factors including NGN2, ISL1, and LHX3, which is necessary and sufficient to induce mature MNs with high efficiencies (~90%) within 3 weeks. This chapter thus provides a robust method to generate high-purity hPSC-MNs at very high yields, enabling the acquisition of rich patient-specific MNs to be used for modeling the molecular underpinnings of MNDs.

Microglia are resident immune cells in the brain and play a central role in the development and surveillance of the nervous system. Extensive gliosis is a common pathological feature of several neurodegenerative diseases, such as Alzheimer's disease (AD), the most common cause of dementia. Microglia can respond to multiple inflammatory insults and later transform into different phenotypes, such as pro- and anti-inflammatory phenotypes, thereby exerting different functions. In recent years, an increasing number of studies based on both traditional bulk sequencing and novel single-cell/nuclear sequencing and multi-omics analysis, have shown that microglial phenotypes are highly heterogeneous and dynamic, depending on the severity and stage of the disease as well as the particular inflammatory milieu. Thus, redirecting microglial activation to beneficial and neuroprotective phenotypes promises to halt the progression of neurodegenerative diseases. To this end, an increasing number of studies have focused on unraveling heterogeneous microglial phenotypes and their underlying molecular mechanisms, including those due to epigenetic and non-coding RNA modulations. In this review, we summarize the epigenetic mechanisms in the form of DNA and histone modifications, as well as the general non-coding RNA regulations that modulate microglial activation during immunopathogenesis of neurodegenerative diseases and discuss promising research approaches in the microglial era.

The CRISPR-based genome editing holds immense potential to fix disease-causing mutations, however, must also handle substantial natural genetic variations between individuals. Previous studies have shown that mismatches between the single guide RNA (sgRNA) and genomic DNA may negatively impact sgRNA efficiencies and lead to imprecise specificity prediction. Hence, the genetic variations bring about a great challenge for designing platinum sgRNAs in large human populations. However, they also provide a promising entry for designing allele-specific sgRNAs for the treatment of each individual. The CRISPR system is rather specific, with the potential ability to discriminate between similar alleles, even based on a single nucleotide difference. Genetic variants contribute to the discrimination capabilities, once they generate a novel protospacer adjacent motif (PAM) site or locate in the seed region near an available PAM. Therefore, it can be leveraged to establish allele-specific targeting in numerous dominant human disorders, by selectively ablating the deleterious alleles. So far, allele-specific CRISPR has been increasingly implemented not only in treating dominantly inherited diseases, but also in research areas such as genome imprinting, haploinsufficiency, spatiotemporal loci imaging and immunocompatible manipulations. In this review, we will describe the working principles of allele-specific genome manipulations by virtue of expanding engineering tools of CRISPR. And then we will review new advances in the versatile applications of allele-specific CRISPR targeting in treating human genetic diseases, as well as in a series of other interesting research areas. Lastly, we will discuss their potential therapeutic utilities and considerations in the era of precision medicine.

Oculocutaneous albinism type 1 (OCA1), resulting from pathogenic variants in the tyrosinase (TYR) gene, refers to a group of phenotypically heterogeneous autosomal recessive disorders characterized by a partial or a complete absence of pigment in the skin/hair and is also associated with common developmental eye defects. In this study, we identified two novel compound heterozygous TYR variants from a Chinese hypopigmentary patient by whole-exome sequencing. Specifically, the two variants were c.-89TG, located at the core of the initiator E-box (Inr E-box) of the TYR promoter, and p.S16Y (c.47CA), located within the signal sequence. We performed both in silico analysis and experimental validation and verified these mutations as OCA1 variants that caused either impaired or complete loss of function of TYR. Mechanistically, the Inr E-box variant dampened TYR binding to microphthalmia-associated transcription factor, a master transcriptional regulator of the melanocyte development, whereas the S16Y variant contributed to endoplasmic reticulum retention, a common and principal cause of impaired TYR activity. Interestingly, we found that the Inr E-box variant creates novel protospacer adjacent motif sites, recognized by nucleases SpCas9 and SaCas9-KKH, respectively, without compromising the functional TYR coding sequence. We further used allele-specific genomic editing by CRISPR activation to specifically target the variant promoter and successfully activated its downstream gene expression, which could lead to potential therapeutic benefits. In conclusion, this study expands the spectrum of TYR variants, especially those within the promoter and noncoding regions, which can facilitate genetic counseling and clinical diagnosis of OCA1.

To investigate the risk factors for serious infections among hospitalized systemic lupus erythematosus (SLE) patients, and to provide the advice for preventing serious infections in SLE patients.Information of SLE patients hospitalized from March 2017 to February 2019 at the Department of Rheumatology and Immunology, Xiangya Hospital, Central South University was obtained. The patients were assigned into a serious infection group and a non-serious infection group. The risk factors for serious infections among SLE inpatients were identified by comparison between the 2 groups and multivariate logistic regression analysis.There were 463 SLE inpatients in total, and 144 were in the serious infection group and 319 in the non-serious infection group. Multivariate logistic regression analysis showed that age ≥54.50 years old (OR=4.958,SLE patients with older age, cardiovascular involvement, hematologic involvement, low serum albumin are prone to suffering serious infections. Increased CRP/ESR ratio indicates serious infections in SLE inpatients. High-dose glucocorticoid and the combined use of immunosuppressive agents can increase the risk of serious infections in SLE inpatients.

Early onset isolated dystonia (DYT1) is a hereditary neurological movement disease caused by a single amino-acid deletion in torsin A (TOR1A), a gene encoding a membrane-embedded ATPase. In this study, we generated an induced pluripotent stem cell (iPSC) line from fibroblasts of a DYT1 patient by the retroviral transduction of Yamanaka factors. The iPSCs retained the heterozygous TOR1A mutation (p.Glu303del), showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers both in vitro and in vivo. This DYT1 patient-specific iPSC will be used for modeling the dystonia pathophysiology and probably drug screening.

Huntington’s disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. The polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTT peptides. To successfully generate functional truncated HTT proteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTT expression. Our results suggest that noncanonical ATGs of N-terminal HTT may not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene.

The flow behavior of sand particles during the core shooting process has been studied by combination of computational fluid dynamics (CFD) simulations and experiments. In the CFD simulations, a two-fluid model (TFM), in which a kinetic-frictional constitutive correlation is incorporated, has been applied to describe the flow dynamics of core shooting process. The influences of turbulence model and boundary conditions on the modeling of core shooting process are further investigated. The simulation results indicate that there are certain sensitivities to the wall boundary condition in the sand flow behavior while no significant difference is observed between the results of the laminar and turbulence models. Using high-speed photography system and pressure measuring system, systematic experiments have been carried out to fully validate the model and good agreement is obtained between simulations and experiments. Based on the simulation and experimental results, the flow behavior of sand particles in the core box is analyzed, and the effects of nozzle dimension (D = 6, 8, 10, 15 mm) and shooting pressure (Pin = 0.3, 0.4, 0.5 MPa) on the hydrodynamic properties such as sand particle mass flux, sand volume fraction at the nozzle and distribution of sand velocity are discussed.

The influence of core sand properties on flow dynamics was investigated synchronously with various core sands, transparent core-box and high-speed camera. To confirm whether the core shooting process has significant turbulence, the flow pattern of sand particles in the shooting head and core box was reproduced with colored core sands. By incorporating the kinetic theory of granular flow (KTGF), kinetic-frictional constitutive correlation and turbulence model, a two-fluid model (TFM) was established to study the flow dynamics of the core shooting process. Two-fluid model (TFM) simulations were then performed and a areasonable agreement was achieved between the simulation and experimental results. Based on the experimental and simulation results, the effects of turbulence, sand density, sand diameter and binder ratio were analyzed in terms of filling process, sand volume fraction (αs) and sand velocity (Vs).

Hydrodynamics of core shooting process is simulated using a computational fluid dynamics model. The model is established based on the Eulerian–Eulerian formulation of a two-fluid model in which a frictional-kinetic constitutive model is incorporated. Both the kinetic stress and the frictional stress are considered in this model. A hyperbolic tangent function is chosen to provide a smooth transition between the kinetic regime and the frictional regime. Experiments are carried out to validate the model and the comparison of the predicted flow behaviour and the pressure curves shows good agreement with the experimental observations. The simulation results indicate that the frictional stress has a significant influence on the flow behaviour of sand and that the frictional-kinetic constitutive model can be well applied to model the core shooting process.

Core shooting process is the most widely used technique to make sand cores and it plays an important role in the quality of sand cores as well as the manufacture of complicated castings in metal casting industry. In this paper, the flow behavior of sand particles in the core box was investigated synchronously with transparent core box, high-speed camera, and pressure measuring system. The flow pattern of sand particles in the shooting head of the core shooting machine was reproduced with various colored core sand layers. Taking both kinetic and frictional stress into account, a kinetic-frictional constitutive correlation was established to describe the internal momentum transfer in the solid phase. Two-fluid model (TFM) simulations with turbulence model were then performed and good agreement was achieved between the experimental and simulation results on the flow behavior of sand particles in both the shooting head and the core box. Based on the experimental and simulation results, the flow behavior of sand particles in the core box, the formation of “dead zone” in the shooting head, and the effect of drag force were analyzed in terms of sand volume fraction (αs), sand velocity (Vs), and pressure variation (P).

Core shooting process plays a decisive role in the quality of sand cores, and core box vents distribution is one of the most important factor determining the effectiveness of core shooting process. In this paper, the influence of core box vents distribution on the flow dynamics of core shooting process was investigated based on in situ experimental observations with transparent core box, high-speed camera and pressure measuring system. Attention was focused on the variation of both the flow behavior of sand and pressure curves due to different vents distribution. Taking both kinetic and frictional stress into account, a kinetic-frictional constitutive model was established to describe the internal momentum transfer in the solid phase. Two-fluid model (TFM) simulation was then performed and good agreement was achieved between the experimental and simulated results on both the flow behavior of sand and the pressure curves. It was found that vents distribution has direct effect on the pressure difference of different locations in the core box, which determines the buoyancy force exerting on the sand particles and significantly influences the filling process of core sand.

Microglia-mediated neuroinflammation is a hallmark of Parkinson's disease (PD). In the brains of patients with PD, microglia have both neurotoxic and neuroprotective effects, depending on their activation state. In this review, we focus on recent research demonstrating the neuroprotective role of microglia in PD. Accumulating evidence indicates that the protective mechanisms of microglia may result from their regulation of transrepression pathways via nuclear receptors, anti-inflammatory responses, neuron-microglia crosstalk, histone modification, and microRNA regulation. All of these mechanisms work together to suppress the production of neurotoxic inflammatory components. However, during the progression of PD, the detrimental effects of inflammation overpower the protective actions of microglia. Therefore, an in-depth exploration of the mechanisms underlying microglial neuroprotection, and a means of promoting the transformation of microglia to the protective phenotype, are urgently needed for the treatment of PD.

In order to optimize the design of core boxes and to improve the quality of cores, it is necessary to understand the behavior of sand particles in core shooting process. For this purpose, a two flu...